Uncontrolled cell growth is the cause of many illnesses in a variety of cell types. For example, cancer occurs when there is an increase in the number of abnormal, or neoplastic, cells derived from a normal tissue that proliferate to form a tumor mass. The tumor cells often invade the adjacent tissues and can spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous growth, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness. Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancel J. Clin. 43:7 (1993)).
Much research has been devoted to discovering new treatments for cell proliferative disorders, such as cancer. Despite recent advances, there is a great need to identify and understand the role of new cellular targets for modulating cell proliferation and to develop alternative or more effective methods of treatment and therapeutic and diagnostic agents. There is also a need to develop alternative therapeutics and methods for treating specific cell types and for treating illnesses caused by or associated with abnormal cell proliferation, such as cancers. For example, desmoplasia is the hyperplasia of fibroblasts and disproportionate formation of fibrous connective tissue, especially in the stroma of carcinomas. Desmoplasia is a hallmark of tumor invasion and malignancy. Desmoid tumors and abdominal fibroids are nodules or relatively large masses of unusually firm scarlike connective tissue resulting from active proliferation of fibroblasts, occurring most frequently in the abdominal muscles of women who have borne children; the fibroblasts infiltrate surrounding muscle and fascia.
In post-natal life, vasculogenesis (endothelial cells forming a primary tubular network) and angiogenesis (the growth or sprouting of new blood vessels from existing vessels) play critical roles in the pathophysiology of neoplastic disorder (Semenza, G. L., (2003) Ann. Rev. Med. 54:17-28). The distinction between vasculogenesis and angiogenesis is not absolute and they overlap (Ribatti, D et al., (2001) Mech. Dev. 100:157-163). Both require endothelial cell proliferation, migration, three-dimensional reorganization of newly formed aggregates and use similar extracellular matrix adhesive mechanisms (Ribatti, supra). Use of anti-angiogenic therapies such as the antibody against vascular endothelial growth (VEGF) called Avastin have been shown to be useful in treating cancers.
Another cellular protein, referred to herein as STOP-1 or UNQ762, has been shown to be overexpressed in certain tumors (e.g., WO 01/163318, WO 01/68848, WO 02/00690, WO 02/08284, WO 02/16602, WO 02/42487). Polyclonal antibodies against STOP-1 have been reported (e.g., WO 02/42487). Although there has been some discussion of targeting STOP-1 to treat cancers and diseases associated with angiogenesis (e.g., WO 01/163318, WO 01/68848, WO 02/00690, WO 02/08284, WO 02/16602, WO 02/42487, WO 00/71581, WO 02/00690), there is a need to further explore the biology of the STOP-1 protein to identify alternative and more effective therapeutic agents and methods for diagnosis and treatment of uncontrolled cell growth and diseases caused by, associated with or complicated by excessive and insufficient angiogenesis.
The present invention addresses these needs and others by providing new STOP-1 polypeptides, antibodies, nucleic acid molecules, compositions and methods that incorporate further knowledge about the STOP-1 protein. Among other things, the present disclosure shows that STOP-1 is overexpressed in the stroma of several tumor types. The present disclosure shows that overexpression of STOP-1 alone can be tumorigenic. Further, the present disclosure demonstrates that the STOP-1 protein can be secreted and that secretion is required for tumorigenesis. Still further, the present disclosure shows that the glycosylation state of STOP-1 affects whether it is secreted and that elimination of a N-glycosylation site, e.g., by substituting the amino acid at position 186 (Asn) with alanine results in loss of secretion. The present disclosure shows that disulfide bonding between STOP-1 proteins can occur at a cysteine 55 in culture in the triple helix domain of STOP-1. Additionally, the present disclosure shows that the STOP-1 protein can form a complex with itself as a dimer, trimer and hexamer and that the c-terminus of the protein is sufficient for oligomerization, whereas a region related to the triple helix domain of collagen is not required. The present disclosure also shows a plurality of agents that specifically bind to STOP-1, including the C-terminal region and N-terminal region of the protein as well as nucleic acid and protein sequences encoding them. Further, the present disclosure shows that STOP-1 expression can be modulated by overexpression of proteins in the WNT signalling pathway that are know to cause breast cancer in mice, e.g., the overexpression of WNT. Additionally, the present disclosure shows that STOP-1 can be cleaved by proteases that are overexpressed in the same tumors as STOP-1, e.g., MMP-9. Further, the present disclosure shows that a method for producing STOP-1 polypeptides by expressing the polypeptides in proteoglycan synthesis deficient cell lines. The present invention shows that STOP-1 binds to the surface of cells, such as cancer cells and endothelial cells. The present invention provides antagonistic molecules that can inhibit the interaction of STOP-1 with the surface of cells. The present invention provides molecules that can potentiate the binding of STOP-1 with the surface of cells. The present invention also relates to the role of STOP-1 in angiogenesis and vasculogenesis and methods and compositions for treating disorders for which treatment would be improved by modulating angiogenesis and vasculogenesis. This data and others provided herein, together with other disclosure of in present application, teach new, better and/or alternative methods for using the STOP-1 protein or compositions relating thereto.